Journal: The Journal of Neuroscience

Article Title: Calpain Inhibition Is Protective in Machado–Joseph Disease Zebrafish Due to Induction of Autophagy

doi: 10.1523/JNEUROSCI.1142-17.2017

Figure Lengend Snippet: MJD pathology and decreased survival of transgenic MJD zebrafish. A, Immunostaining for polyglutamine and ataxin-3 (antibody recognized both endogenous zebrafish ataxin-3 and transgenic human ataxin-3) in sections throughout the medulla of 12-month-old zebrafish revealed polyglutamine-positive neuritic beading pattern (arrows) in EGFP-ataxin-3-84Q zebrafish only. This neuritic beading pattern was also positive for ataxin-3. Scale bar, 35 μm. B, Quantification of the number of neurites showing the polyglutamine-positive neuritic beading pattern within the medulla region revealed that the phenotype was only present in EGFP-ataxin-3-84Q zebrafish sections (n = 6 each, ***p < 0.0001). C, Quantification of the number of neurites showing ataxin-3-positive beading pattern revealed that it was far more common in EGFP-ataxin-3-84Q zebrafish than nontransgenic and EGFP-ataxin-3-23Q (n = 6 each, **p < 0.001). D, The lifespan of nontransgenic (n = 15), EGFP-ataxin-3-23Q (n = 16) and EGFP-ataxin-3-84Q (n = 16) zebrafish is plotted within a Kaplan–Meier survival graph that reveals significantly different survivals for the three groups, p < 0.0279. Error bars represent mean ± SEM.

Article Snippet: Antibodies used included rabbit anti-MJD (kind gift from H. Paulson), rabbit anti-beclin-1 (11306-1-AP, Proteintech), rabbit anti-p62 (SQSTM1, MBL PM045, MBL Life Science), rabbit anti-LC3B (ab51520, Abcam), mouse anti-spectrin (sc-48382, Santa Cruz Biotechnology), mouse anti-β-actin (A5441, Sigma-Aldrich) and mouse anti-GAPDH (60004-1, Proteintech) for loading controls.

Techniques: Transgenic Assay, Immunostaining

Journal: The Journal of Neuroscience

Article Title: Calpain Inhibition Is Protective in Machado–Joseph Disease Zebrafish Due to Induction of Autophagy

doi: 10.1523/JNEUROSCI.1142-17.2017

Figure Lengend Snippet: Calpeptin treatment rescues the motor phenotype of MJD zebrafish at 6 dpf, concurrent with a decrease in human ataxin-3 protein levels and induction of autophagy. A, The total distance swum during a 4 min darkness response test is shown for DMSO-treated nontransgenic (n = 69), EGFP-ataxin-3-23Q (n = 68), and EGFP-ataxin-3-84Q larva (n = 151), and EGFP-ataxin-3-84Q larva treated with increasing concentrations of calpeptin (6.25–100 μm: n = 26–105). DMSO-treated EGFP-ataxin-3-84Q swam significantly shorter distances than the DMSO-treated nontransgenic and EGFP-ataxin-3-23Q larva (*significant p < 0.023). EGFP-ataxin-3-84Q larva treated with 25–100 μm Calpeptin swam significantly longer distances compared with their DMSO-treated siblings; ***p < 0.001. B, Immunoblots of calpeptin-treated MJD zebrafish revealed that calpeptin treatment decreased levels of ataxin-3 in a dose-dependent manner. Probing the immunoblot for beclin-1, LC3-I/II, and p62 revealed that calpeptin also decreased levels of p62 and beclin-1 increased the amount of LC3-II. C, Quantification of band intensity of ataxin-3 cleavage fragment levels from separate immunoblots (n = 3–8) revealed a dose-dependent decrease, with a significant decrease following treatment with 50 and 100 μm calpeptin compared with DMSO-treated EGFP-ataxin-3-84Q zebrafish (**p < 0.008). D, Quantification of full-length ataxin-3 levels revealed a significant decrease in full-length ataxin-3 levels following treatment with 25–100 μm calpeptin compared with DMSO EGFP-ataxin-3-84Q (**p < 0.01). E, Quantification of the intensity of the p62 band revealed that a significant decrease in p62 levels following treatment with 25–100 μm calpeptin compared with DMSO-treated EGFP-ataxin-3-84Q, **p < 0.01, n = 3–8. F, ELISA assay of 6 dpf protein lysates confirmed that calpeptin treatment (50 μm) did significantly reduce levels of human ataxin-3 in EGFP-ataxin-3-84Q zebrafish compared with DMSO treatment (n = 4–5, *p = 0.036). FL, Full-length; CF, cleavage fragment; ZF, zebrafish. Error bars represent mean ± SEM.

Article Snippet: Antibodies used included rabbit anti-MJD (kind gift from H. Paulson), rabbit anti-beclin-1 (11306-1-AP, Proteintech), rabbit anti-p62 (SQSTM1, MBL PM045, MBL Life Science), rabbit anti-LC3B (ab51520, Abcam), mouse anti-spectrin (sc-48382, Santa Cruz Biotechnology), mouse anti-β-actin (A5441, Sigma-Aldrich) and mouse anti-GAPDH (60004-1, Proteintech) for loading controls.

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Neuroscience

Article Title: Calpain Inhibition Is Protective in Machado–Joseph Disease Zebrafish Due to Induction of Autophagy

doi: 10.1523/JNEUROSCI.1142-17.2017

Figure Lengend Snippet: Real-time PCR revealed that changes in human ataxin-3 and p62 levels following calpeptin treatment were not present at an mRNA level. A, Real-time PCR of human ATXN3 mRNA levels in control (nontransgenic, n = 3), EGFP-ataxin-3-23Q (n = 3) and EGFP-ataxin-3-84Q (with and without 50 μm calpeptin treatment, n = 3 and 4, respectively) revealed similar human ataxin-3 levels in all transgenic MJD zebrafish samples. All transgenic ataxin-3 zebrafish groups had significantly higher human ATXN3 levels than control (∧p < 0.041), with no significant decrease in human ATXN3 mRNA levels in EGFP-ataxin-3-84Q larva receiving calpeptin (50 μm) treatment. B, Real-time PCR of p62 mRNA levels in control, EGFP-ataxin-3-23Q and EGFP-ataxin-3-84Q (with and without calpeptin treatment) samples revealed that EGFP-ataxin-3-84Q zebrafish have higher p62 mRNA levels than control and EGFP-ataxin-3-23Q larva; #p < 0.031. Calpeptin treatment did not effect the p62 mRNA levels of EGFP-ataxin-3-84Q zebrafish. n values were the same as in A. Error bars represent mean ± SEM.

Article Snippet: Antibodies used included rabbit anti-MJD (kind gift from H. Paulson), rabbit anti-beclin-1 (11306-1-AP, Proteintech), rabbit anti-p62 (SQSTM1, MBL PM045, MBL Life Science), rabbit anti-LC3B (ab51520, Abcam), mouse anti-spectrin (sc-48382, Santa Cruz Biotechnology), mouse anti-β-actin (A5441, Sigma-Aldrich) and mouse anti-GAPDH (60004-1, Proteintech) for loading controls.

Techniques: Real-time Polymerase Chain Reaction, Transgenic Assay

Journal: American Journal of Translational Research

Article Title: LncRNA MALAT1 promotes breast cancer progression by sponging miR101-3p to mediate mTOR/PKM2 signal transmission

doi:

Figure Lengend Snippet: Sequences of all the primers utilized in this study

Article Snippet: All the primary antibodies used were rabbit anti-β-actin polyclonal antibodies (pab, 1:2000, ProteinTech, Wuhan, China), rabbit anti-mTOR, and anti-PKM2 pab (1:2000, CST, MA, USA).

Techniques:

Journal: American Journal of Translational Research

Article Title: LncRNA MALAT1 promotes breast cancer progression by sponging miR101-3p to mediate mTOR/PKM2 signal transmission

doi:

Figure Lengend Snippet: MALAT1 knockdown inhibits the mTOR/PKM2 pathway. (A) On the basis of mutating the miR-101-3p candidate binding site in mTOR, the luciferase reporter plasmids of mTOR-Mut and mTOR-WT were cloned. (B) BC cells cotransfected with luciferase reporter plasmids and NC or miR-101-3p mimics, and luciferase activities measured using dual-luciferase assays. (C) BC cells transfected with the NC or miR-101-3p mimics and assessing the mTOR expression in the cells using qRT-PCR. (D, E) pcDNA3.1-NC or pcDNA3. MALAT1 transfected into BC cells, and MALAT1 and miR-101-3p expression in the cells analyzed using qRT-PCR. (F) mTOR/PKM2 pathway expression performed using Western blot (G) Association of the MALAT1 level with the mTOR expression in the BC clinical samples identified using Spearman’s correlation. (H) The mTOR/PKM2 pathway level in the BC cells assessed using Western blot, and the cells cotransfected with si-MALAT1#2, or si-MALAT1#2 and NC or an miR-101-3p inhibitor. Compared to the NC or OE-NC group, **P < 0.01, ***P < 0.001.

Article Snippet: All the primary antibodies used were rabbit anti-β-actin polyclonal antibodies (pab, 1:2000, ProteinTech, Wuhan, China), rabbit anti-mTOR, and anti-PKM2 pab (1:2000, CST, MA, USA).

Techniques: Binding Assay, Luciferase, Clone Assay, Transfection, Expressing, Quantitative RT-PCR, Western Blot